Pharmaceutical formulations of nitrite and uses thereof

ABSTRACT

The present invention relates to pharmaceutical compositions of nitrites such as inorganic nitrites, or any pharmaceutically acceptable salts, solvates, or prodrugs thereof, and the medical use of these compositions. The pharmaceutical compositions, which can be formulated for oral administration, can provide immediate release or extended release of the nitrite ion (NO2−). The pharmaceutical compositions of the invention are useful, for example, for treating or reducing pain, improving symptoms of a microvascular disease, and improving nerve conduction velocity.

FIELD OF THE INVENTION

The invention features pharmaceutical compositions of nitrites andmethods using these compositions to treat or reduce pain and symptoms ofmicrovascular disease.

BACKGROUND

Nitric oxide (NO) is involved in many physiological processes and playsa key role in redox signaling. In particular, endothelial-derived NOregulates normal vascular function by stimulating NO-dependentactivation of soluble guanylate cyclase, which leads to the activationof a signaling cascade resulting in smooth muscle relaxation andvasodilation. Dysfunction in NO-dependent signaling processes can occureither through a deficit in NO synthesis, NO bioavailability, or both.For instance, studies have shown that endothelial-derived NO productionis reduced in patients with peripheral artery disease. Thus, reduced NObioavailability might substantially contribute to the development ofmicrovascular disease.

Studies have also indicated that NO plays a complex and diverse role inthe incidence of pain. For instance, NO is an essential neurotransmitterinvolved in the nociceptive process and contributes to centralsensitization in the dorsal horn of the spinal cord. In contrast, otherstudies have implicated that NO can inhibit nociception in the centraland peripheral nervous system. NO has also been shown to mediate theanalgesic effect of opioids, such as morphine.

Accordingly, there exists a need in the medical field to develop safeand effective treatments that restore NO bioavailability in patientswith microvascular disease and that provide a source of NO to mitigatepain. Thus, therapeutic strategies that modulate NO are highlydesirable.

SUMMARY OF THE INVENTION

The invention features methods to treat or reduce pain in a patient(such as a mammal (e.g., a human)) by administering sodium nitrite or apharmaceutically acceptably salt thereof. Additionally, methods oftreating a microvascular disease (e.g., peripheral artery disease,diabetic neuropathy, scleroderma, Raynaud's disease, cerebral autosomaldominant arteriopathy with sub-cortical infarcts andleukoencephalopathy, thrombotic microangiopathy, or thromboangiitisobliterans) by administering sodium nitrite or a pharmaceuticallyacceptably salt thereof to a patient are disclosed. The methods featurea first step of administering a low dose (e.g., about 5 mg to about 50mg twice daily) of inorganic nitrite to the patient to, e.g., treat orreduce pain. The methods feature a second step of administering a highdose (e.g., about 60 mg to about 100 mg twice daily) of inorganicnitrite to the patient to, e.g., treat symptoms of microvasculardisease. The invention also feature methods of improving nerveconduction velocity in a patient (e.g., a patient identified as being inneed of improved nerve conduction velocity) by administering a high dose(e.g., about 60 mg to about 100 mg (e.g., 80 mg) twice daily) ofinorganic nitrite to the patient to increase nerve conduction velocity.

A first aspect of the invention features a method of treating orreducing pain, such as chronic pain, in a patient (such as a mammal(e.g., a human)). The method of the first aspect includes: (i)administering about 5 mg to about 50 mg of inorganic nitrite or apharmaceutically acceptable salt thereof (e.g., 40 mg of inorganicnitrite or a pharmaceutically acceptable salt thereof) to the patienttwo times per day for a first treatment period of 6 weeks to 14 weeks(e.g., at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10weeks, at least 11 weeks, at least 12 weeks, or at least 13 weeks); andthen (ii) administering about 60 mg to about 100 mg of inorganic nitriteor a pharmaceutically acceptable salt thereof (e.g., 80 mg of inorganicnitrite or a pharmaceutically acceptable salt thereof) to the patienttwo times per day for a second treatment period of 6 weeks to 14 weeks(e.g., at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10weeks, at least 11 weeks, at least 12 weeks, or at least 13 weeks).Preferably, the first treatment period is at least 10 weeks.

In some embodiments, the pain is neuropathic pain, inflammatory pain,nociceptive pain, functional pain, musculo-skeletal pain, or centralnervous system pain. In some embodiments, the neuropathic pain isdiabetic peripheral neuropathy, post-herpetic neuralgia, trigeminalneuralgia, phantom limb pain, carpal tunnel syndrome, sciatica, pudendalneuralgia, complex regional pain syndrome, sensory polyneuropathies,mono-neuropathies, or central pain syndrome.

Additionally, in some embodiments, the patient can have a microvasculardisease, such as peripheral artery disease, diabetic neuropathy,scleroderma, Raynaud's disease, cerebral autosomal dominant arteriopathywith sub-cortical infarcts and leukoencephalopathy, thromboticmicroangiopathy, or thromboangiitis obliterans. In some embodiments, thepatient can also have type 1 diabetes or type 2 diabetes.

A second aspect of the invention features a method of treating amicrovascular disease in a patient (such as a mammal (e.g., a human)).For example, the microvascular disease is peripheral artery disease,diabetic neuropathy, scleroderma, Raynaud's disease, cerebral autosomaldominant arteriopathy with sub-cortical infarcts andleukoencephalopathy, thrombotic microangiopathy, or thromboangiitisobliterans. In some embodiments, the patient can have type 1 diabetes ortype 2 diabetes.

The method of the second aspect includes the steps of: (i) administeringabout 5 mg to about 50 mg of inorganic nitrite or a pharmaceuticallyacceptable salt thereof (e.g., 40 mg of inorganic nitrite or apharmaceutically acceptable salt thereof) to the patient two times perday for a first treatment period of 6 weeks to 14 weeks (e.g., at least7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least11 weeks, at least 12 weeks, or at least 13 weeks); and then (ii)administering about 60 mg to about 100 mg (e.g., 80 mg of inorganicnitrite or a pharmaceutically acceptable salt thereof) of inorganicnitrite or a pharmaceutically acceptable salt thereof to the patient twotimes per day for a second treatment period of 6 weeks to 14 weeks(e.g., at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10weeks, at least 11 weeks, at least 12 weeks, or at least 13 weeks).Preferably, the first treatment period is at least 10 weeks.

The method can optionally further include determining whether thepatient, such as a patient with chronic pain, exhibits a reduction inpain. In some embodiments, the reduction in pain is determined as adecrease in pain intensity, frequency, duration, and/or improvements inquality of life. For example, the method can optionally includeperforming a Brief Pain Inventory, a Neuropathic Pain Symptom Inventory,and/or a McGill Pain Questionnaire to determine if the patient exhibitsa reduction in pain. Desirably, step (i) of the method results in areduction in pain. In some embodiments, step (ii) is performed when thepatient exhibits a reduction in pain.

The method can also include the optional step of determining whether thepatient (such as a mammal (e.g., a human)) experiences an improvement inone or more symptoms of the microvascular disease, e.g., in which theimprovement in one or more symptoms of the microvascular disease isdetermined as an increase in nitric oxide availability, physicalfunction, and/or motor neuron activity. For example, the method canoptionally include performing flow-mediated dilation to determine nitricoxide availability, a RAND-36 Questionnaire to determine physicalfunction, and/or an assessment of nerve conduction velocity to determinemotor neuron activity. Preferably, step (ii) of the method results in animprovement in one or more symptoms of the microvascular disease.

A third aspect of the invention features a method of improving nerveconduction velocity in a patient (e.g., a mammal, such as a human)identified as being in need of improved nerve conduction velocity. Themethod of the third aspect includes administering about 60 mg to about100 mg of inorganic nitrite or a pharmaceutically acceptable saltthereof twice daily (e.g., about 70 mg to about 90 mg twice daily, suchas about 70 mg, about 75 mg, or about 80 mg twice daily) to the patientfor a treatment period (e.g, at least 6 weeks, at least 7 weeks, atleast 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks,at least 12 weeks, at least 13 weeks, or at least 14 weeks or longer)sufficient to improve nerve conduction velocity in the patient.

In some embodiments, the method of the third aspect further includesmonitoring whether there is an increase in nerve conduction velocity inthe patient (e.g., a mammal, such as a human), such as by performing anerve conduction velocity test and/or a sensory test to assess thepatient's ability to feel, e.g., pain. For example, there is an increasein nerve conduction velocity after administration of the inorganicnitrite or the pharmaceutically acceptable salt (e.g., an increase innerve conduction velocity of about 1 m/s, about 1.5 m/s, about 2 m/s,about 2.5 m/s, about 3 m/s, about 3.5 m/s, about 4 m/s, about 4.5 m/s,about 5 m/s, about 5.5 m/s, about 6 m/s, or about 6.5 m/s or more)relative to the nerve conduction velocity of the patient prior toadministration of the inorganic nitrite or the pharmaceuticallyacceptable salt. In some embodiments, a sensory test may be performedusing, e.g., a monofilament, placed on the patient's skin, such as onthe patient's feet, to assess the nerve conduction velocity of thepatient. For example, the patient exhibits an increased ability to sensethe monofilament after administration of the inorganic nitrite (e.g.,about 60 mg to about 100 mg of inorganic nitrite twice daily, such asabout 80 mg twice daily) relative to the ability of the patient to sensethe monofilament prior to administration of the inorganic nitrite.

In some embodiments, the patient (e.g., a mammal, such as a human) has amicrovascular disease, such as diabetic neuropathy, peripheral arterydisease, scleroderma, Raynaud's disease, cerebral autosomal dominantarteriopathy with sub-cortical infarcts and leukoencephalopathy,thrombotic microangiopathy, or thromboangiitis obliterans. For example,the method of the third aspect further includes determining whether thepatient experiences an improvement in one or more symptoms of themicrovascular disease, e.g., in which the improvement in one or moresymptoms of the microvascular disease is determined as an increase innitric oxide availability or physical function. In preferredembodiments, the patient experiences an improvement in one or moresymptoms of the microvascular disease after administration of theinorganic nitrite or the pharmaceutically acceptable salt. Additionally,in some embodiments, the patient does not have pain associated with themicrovascular disease.

In any of the above aspects, the inorganic nitrite can be, e.g., NaNO₂,KNO₂, or Ca(NO₂)₂. Preferably, the inorganic nitrite is NaNO₂. Forexample, the inorganic nitrite or the pharmaceutically acceptable saltthereof can be formulated, e.g., for topical, enteral, or parenteraladministration and/or as a solid dosage form for oral administration. Insome embodiments, the inorganic nitrite or the pharmaceuticallyacceptable salt thereof is formulated as a tablet or capsule, such asfor sustained release of the inorganic nitrite. In some embodiments, theinorganic nitrite or the pharmaceutically acceptable salt thereof can beformulated with one or more pharmaceutically acceptable excipients.

Definitions

As used herein, “a” or “an” means “at least one” or “one or more” unlessotherwise indicated. In addition, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to a composition containing “atherapeutic agent” includes a mixture of two or more therapeutic agents.

As used herein, “about” refers to an amount that is ±10% of the recitedvalue and is preferably +5% of the recited value, or more preferably ±2%of the recited value.

As used herein, “at least” refers to an amount that is ≤10% of therecited value and is preferably ≤5% of the recited value, or morepreferably ≤2% of the recited value.

By “chronic pain” is meant pain that lasts longer than three to sixmonths or pain that extend beyond the expected period of healing.Chronic pain may originate with an initial trauma/injury or infection,or may be an ongoing cause of pain associated with neuropathic pain(e.g., diabetic peripheral neuropathy, post-herpetic neuralgia,trigeminal neuralgia, phantom limb pain, carpal tunnel syndrome,sciatica, pudendal neuralgia, complex regional pain syndrome, sensorypolyneuropathies, mono-neuropathies, or central pain syndrome),headaches, joint pain, backaches, sinus pain, muscle pain, nerve pain,and pain affecting specific parts of the body, such as shoulders,pelvis, and neck. Chronic pain may also be associated with lower backpain, arthritis, headache, multiple sclerosis, fibromyalgia, shingles,nerve damage, or cancer.

As used interchangeably herein, the terms “decreasing” and “reducing”refer to the ability to cause an overall decrease preferably of 20% orgreater, more preferably of 50% or greater, and most preferably of 75%,85%, 90%, 95%, or greater. In particular, decreasing or reducing canrefer to treatment that alleviates one or more symptoms of a disease,disorder, or conditions described herein (e.g., a microvascular disorderor pain).

As used herein, the term “delayed release” refers to a pharmaceuticalpreparation, e.g., an orally administered formulation of inorganicnitrite, which passes through the stomach substantially intact anddissolves in the small and/or large intestine (e.g., the colon). Forinstance, delayed release of the active agent (e.g., inorganic nitriteas described herein) results from the use of an enteric coating of anoral medication (e.g., an oral dosage form).

The term an “effective amount” of an agent, such as inorganic nitrite,is the amount sufficient to effect beneficial or desired results, suchas clinical results, and, as such, an “effective amount” depends uponthe context in which it is being applied.

The terms “extended release” or “sustained release” interchangeablyrefer to a drug formulation that provides for gradual release of a drug(e.g., inorganic nitrite) over an extended period of time, e.g., 6-12hours or more, compared to an immediate release formulation of the samedrug. Preferably, although not necessarily, the formulation results insubstantially constant blood levels of a drug over an extended timeperiod that are within therapeutic levels and fall within a peak plasmaconcentration range that is between, for example, 0.05-10 μM, 0.1-10 μM,0.1-5.0 μM, or 0.1-1 μM.

As used herein, the term “microvascular disease” refers to diseases ordisorders of small, resistance vessels (e.g., pre-capillary arterioles)with an internal diameter of less than 100 microns, which can be causedby metabolic or oxidative stress leading to microvascular dysfunctionand/or damage. For example, microvascular disease includes, but is notlimited to, peripheral artery disease, diabetic neuropathy, scleroderma,Raynaud's disease, cerebral autosomal dominant arteriopathy withsub-cortical infarcts and leukoencephalopathy, thromboticmicroangiopathy, or thromboangiitis obliterans.

As used herein, the term “monofilament” refers to a plastic fiber usedby, e.g., a physician, to assess a patient's ability to sense pressureon their extremities, such as the feet, in a monofilament test. Themonofilament test, for example, is administered to a patient to assessthe nerve conduction velocity of the patient and/or to determine if thepatient has a disorder associated with decreased nerve conductionvelocity (e.g., a microvascular disease, such as diabetic neuropathy).The bent monofilament may exert a pressure of, e.g., 10 grams, on thepatient's foot. During a monofilament test, the bent or unbentmonofilament is touched to different points on the sole of the patient'sfoot. The patient will then identify when they sense the monofilament.If the patient is unable to sense the bent monofilament, e.g., exertinga pressure of 10 grams on the patient's foot, then the patient ischaracterized as being in need of improved nerve conduction velocityand/or may have a microvascular disease, such as diabetic neuropathy,peripheral artery disease, scleroderma, Raynaud's disease, cerebralautosomal dominant arteriopathy with sub-cortical infarcts andleukoencephalopathy, thrombotic microangiopathy, or thromboangiitisobliterans. The monofilament test allows for the identification ofpatients in need of improved nerve conduction velocity and themonitoring of patients for improvements in nerve conduction velocityafter administration of inorganic nitrite as described herein.

As used herein, the term “neuropathic pain” refers to pain caused bydamage or disease affecting the somatosensory nervous system. Forexample, neuropathic pain includes, but is not limited to, diabeticperipheral neuropathy, post-herpetic neuralgia, trigeminal neuralgia,phantom limb pain, carpal tunnel syndrome, sciatica, pudendal neuralgia,complex regional pain syndrome, sensory polyneuropathies,mono-neuropathies, or central pain syndrome, headaches, joint pain,backaches, sinus pain, muscle pain, nerve pain, and pain affectingspecific parts of the body, such as shoulders, pelvis, and neck, and/orpain that is associated with lower back pain, arthritis, headache,multiple sclerosis, fibromyalgia, shingles, nerve damage, or cancer.

As used herein, the term “nerve conduction velocity” refers to the speedof one or more electrochemical signals across a neural pathway.Measurements of nerve conduction velocity serve as an indicator of nervehealth in patients. For example, nerve conduction velocity tests may beperformed by, e.g., attaching two electrode's to a patient's skin over atest nerve, administering an electrical impulse through one electrode tostimulate the nerve, recording the electrical impulse through the nerveat the second electrode, and determining the time difference betweenstimulation from the first to second electrode (e.g., in m/s). Nerveconduction velocity tests may also be performed using a multi-electrodearray (e.g., a 3-dimensional electrode array).

As used herein, “prevention” refers to a prophylactic treatment, such asinorganic nitrite, given to a subject who has or will have a disease, adisorder, a condition (e.g., pain), or one or more symptoms associatedwith a disease, a disorder, or a condition (e.g., symptoms associatedwith microvascular disease).

The term “pharmaceutical composition,” as used herein, represents acomposition containing a compound described herein (e.g., inorganicnitrite, or any pharmaceutically acceptable salt, solvate, or prodrugthereof), formulated with a pharmaceutically acceptable excipient, andtypically manufactured or sold with the approval of a governmentalregulatory agency as part of a therapeutic regimen for the treatment ofdisease in a mammal. Pharmaceutical compositions can be formulated, forexample, for oral administration in unit dosage form (e.g., a tablet,capsule, caplet, gelcap, or syrup); for topical administration (e.g., asa cream, gel, lotion, or ointment); for intravenous administration(e.g., as a sterile solution free of particulate emboli and in a solventsystem suitable for intravenous use); or in any other formulationdescribed herein.

A “pharmaceutically acceptable excipient,” as used herein, refers to anyingredient other than the compounds described herein (for example, avehicle capable of suspending or dissolving the active compound) andhaving the properties of being nontoxic and non-inflammatory in apatient. Excipients may include, for example, antiadherents,antioxidants, binders, coatings, compression aids, disintegrants, dyes(colors), emollients, emulsifiers, fillers (diluents), film formers orcoatings, flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, or waters of hydration. Exemplary excipients include, butare not limited to, butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, maltose,mannitol, methionine, methylcellulose, methyl paraben, microcrystallinecellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone,pregelatinized starch, propyl paraben, retinyl palmitate, shellac,silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodiumstarch glycolate, sorbitol, starch (corn), stearic acid, stearic acid,sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, andxylitol.

The term “pharmaceutically acceptable salt,” as use herein, representsthose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example,pharmaceutically acceptable salts are described in: Berge et al., J.Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts:Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth),Wiley-VCH, 2008. The salts can be prepared in situ during the finalisolation and purification of the compounds of the invention orseparately by reacting the free base group with a suitable organic orinorganic acid. Representative acid addition salts include acetate,adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, orethylamine.

As used interchangeably herein, the terms “subject” and “patient” referto any animal (such as a mammal, e.g., a human). A subject to be treatedor tested for responsiveness to a therapy according to the methodsdescribed herein can be one who has been diagnosed with pain and/or amicrovascular disease.

As used herein, “treating” refers to administering a pharmaceuticalcomposition, such as inorganic nitrite, for prophylactic and/ortherapeutic purposes. To “reduce the likelihood” refers to prophylactictreatment of a patient who is not yet ill, but who is susceptible to, orotherwise at risk of, a particular disease or condition (e.g., theconditions described herein, such as pain and/or microvascular disease).To “treat disease” or use for “therapeutic treatment” refers toadministering treatment to a patient already suffering from a disease toameliorate the disease and improve the patient's condition. The term“treating” also includes treating a patient to delay progression of adisease or its symptoms. Beneficial or desired results can include, butare not limited to, alleviation, amelioration, or prevention of painand/or microvascular disease, a condition associated with pain and/ormicrovascular disease, or one or more symptoms associated with painand/or microvascular disease.

The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with anysuitable pharmaceutical excipient or excipients.

The recitation herein of numerical ranges by endpoints is intended toinclude all numbers subsumed within that range (e.g., a recitation of 1to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

Other features and advantages of the invention will be apparent from thefollowing Detailed Description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing flow mediated dilation (FMD) results for PADpatients at baseline and after 10 weeks of receiving placebo, 40 mg ofan immediate release formulation of sodium nitrite (TV1001) twice daily(BID), or 80 mg TV1001 BID. The results are presented in adjusted leastsquare means and as the difference in percentage change from baselineFMD.

FIG. 2 is a graph showing RAND-36 Questionnaire results for PAD patientsat baseline and after 10 weeks of receiving placebo, 40 mg TV1001 BID,or 80 mg TV1001 BID. Patients were evaluated in the RAND-36Questionnaire categories of Physical Functioning, Limitations due toPhysical Health, Energy/Fatigue, Improvements in Pain, and GeneralHealth.

FIG. 3 is a graph showing daily pain logs for patients with diabeticneuropathy over 86 days of treatment with 40 mg TV1001sr BID, 80 mgTV1001sr BID, or placebo.

FIG. 4 is a graph showing nerve conduction velocity for patients withdiabetic neuropathy at baseline (Visit 1) and after 10 weeks (Visit 3)of receiving 40 mg of a sustained release formulation of sodium nitrite(TV1001sr) BID, 80 mg TV1001sr BID, or placebo.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered that inorganic nitrite (e.g., sodium nitrite (NaNO₂))can be used to effectively treat both pain and symptoms associated withmicrovascular disease in a patient (e.g., a human). In particular,administration of inorganic nitrite at relatively low doses (e.g., about5 mg to about 50 mg of inorganic nitrite twice daily) significantlyreduces pain, while administration of inorganic nitrite at relativelyhigh doses (e.g., 60 mg to about 100 mg of inorganic nitrite twicedaily) treats symptoms associated with microvascular disease, such asnitric oxide availability, physical function, and motor neuron activity.Additionally, administration of about 60 mg to about 100 mg of inorganicnitrite twice daily (e.g., about 80 mg twice daily) improves nerveconduction velocity in patients identified as being in need of improvednerve conduction velocity (e.g., patients having a microvasculardisease, such as diabetic neuropathy, peripheral artery disease,scleroderma, Raynaud's disease, cerebral autosomal dominant arteriopathywith sub-cortical infarcts and leukoencephalopathy, thromboticmicroangiopathy, or thromboangiitis obliterans). In particular, patientscan be identified as being in need of improved nerve conduction velocityusing a monofilament prior to administration of the inorganic nitrite.

For example, the methods featuring the administration of inorganicnitrite can be used to reduce pain, such as chronic pain, in patientswith microvascular disease (e.g., peripheral artery disease, diabeticneuropathy, scleroderma, Raynaud's disease, cerebral autosomal dominantarteriopathy with sub-cortical infarcts and leukoencephalopathy,thrombotic microangiopathy, or thromboangiitis obliterans).Additionally, inorganic nitrite can be used to treat or reduceneuropathic pain (e.g., diabetic peripheral neuropathy, post-herpeticneuralgia, trigeminal neuralgia, phantom limb pain, carpal tunnelsyndrome, sciatica, pudendal neuralgia, complex regional pain syndrome,sensory polyneuropathy, mono-neuropathies, and central pain syndrome).

Inorganic nitrite (e.g., sodium nitrite (NaNO₂)) can also be used toimprove nerve conduction velocity in a patient (e.g, a patientidentified as being in need of improved nerve conduction velocity). Inparticular, administration of inorganic nitrite can improve nerveconduction velocity in patients with microvascular diseases, such asperipheral artery disease, diabetic neuropathy, scleroderma, Raynaud'sdisease, cerebral autosomal dominant arteriopathy with sub-corticalinfarcts and leukoencephalopathy, thrombotic microangiopathy, andthromboangiitis obliterans. Additionally, these patients may not havepain associated with a microvascular disease.

Inorganic Nitrite

The compositions of the invention include inorganic nitrite, e.g., asalt or ester of nitrous acid (HNO₂), or a pharmaceutically acceptablesalt thereof. In particular, inorganic nitrite, such as NaNO₂, KNO₂, orCa(NO₂)₂, can be used to reduce pain in a patient, e.g., a patient thathas a microvascular disease, or to treat symptoms of microvasculardisease (e.g., peripheral artery disease, diabetic neuropathy,scleroderma, Raynaud's disease, cerebral autosomal dominant arteriopathywith sub-cortical infarcts and leukoencephalopathy, thromboticmicroangiopathy, and thromboangiitis obliterans). Inorganic nitrite,such as NaNO₂, KNO₂, or Ca(NO₂)₂, can also be used to improve nerveconduction velocity in a patient (e.g., a patient with a microvasculardisease, such as diabetic neuropathy or peripheral artery disease)identified as being in need of improved nerve conduction velocity, suchas by increasing nerve conduction velocity in the patient.

Given the results described herein, the treatment methods are notlimited to administration of a particular form of inorganic nitrite.Nitrite salts that can be used in the methods include, withoutlimitation, salts of alkali metals, e.g., sodium, potassium; salts ofalkaline earth metals, e.g., calcium, magnesium, and barium; and saltsof organic bases, e.g., amine bases and inorganic bases. Compounds thatcan be used in the methods featuring inorganic nitrite also include allisotopes of atoms occurring in the intermediate or final compounds.Isotopes include those atoms having the same atomic number but differentmass numbers. For example, isotopes of hydrogen include tritium anddeuterium. Inorganic nitrite compounds are also meant to includesolvated (e.g., hydrated) forms. Nitrite has the chemical formula NO₂ ⁻and may exist as an ion in water. Sodium nitrite has the chemicalformula NaNO₂ and typically dissolves in water to form the sodium ionNa⁺ and the nitrite ion NO₂ ⁻. Exemplary nitrite compounds are describedin WO 2008/105730, hereby incorporated by reference in its entirety.

Representative inorganic nitrite compounds that can be used according tothe methods for treating pain and/or symptoms of microvascular diseaseinclude ammonium nitrite (NH₄NO₂), barium nitrite (Ba(NO₂)₂; e.g.,anhydrous barium nitrite or barium nitrite monohydrate), calcium nitrite(Ca(NO₂)₂; e.g., anhydrous calcium nitrite or calcium nitritemonohydrate), cesium nitrite (CsNO₂), cobalt(II) nitrite (Co(NO₂)₂),cobalt(III) potassium nitrite (CoK₃(NO₂)₆; e.g., cobalt(III) potassiumnitrite sesquihydrate), lithium nitrite (LiNO₂; e.g., anhydrous lithiumnitrite or lithium nitrite monohydrate), magnesium nitrite (MgNO₂; e.g.,magnesium nitrite trihydrate), postassium nitrite (KNO₂), rubidiumnitrite (RbNO₂), silver (I) nitrite (AgNO₂), strontium nitrite(Sr(NO₂)₂), and zinc nitrite (Zn(NO₂)₂).

Inorganic nitrite compounds that are useful in the methods of treatingpain and/or microvascular disease can be prepared in a variety of waysknown to one of ordinary skill in the art of chemical synthesis. Methodsfor preparing nitrite salts are well known in the art and a wide rangeof precursors and nitrite salts are readily available commercially.Nitrites of the alkali and alkaline earth metals can be synthesized byreacting a mixture of nitrogen monoxide (NO) and nitrogen dioxide (NO₂)with a corresponding metal hydroxide solution, as well as through thethermal decomposition of the corresponding nitrate. Other nitrites areavailable through the reduction of the corresponding nitrates.

Suitable pharmaceutically acceptable salts for use in the methods oftreating pain and/or microvascular disease include, for example, sodiumnitrite, potassium nitrite, or calcium nitrite. Still other exemplarysalts are found in Remington's Pharmaceutical Sciences, 17th ed., MackPublishing Company, Easton, Pa., 1985, p. 1418, Berge et al., J.Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts:Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth),Wiley-VCH, 2008, each of which is incorporated herein by reference inits entirety.

Pharmaceutical Compositions

Inorganic nitrite, e.g., a salt of nitrous acid (HNO₂) such as NaNO₂, ora pharmaceutically acceptable salt can be administered in the form ofpharmaceutical compositions to reduce pain and to treat the symptoms ofmicrovascular disease (e.g., peripheral artery disease, diabeticneuropathy, scleroderma, Raynaud's disease, cerebral autosomal dominantarteriopathy with sub-cortical infarcts and leukoencephalopathy,thrombotic microangiopathy, or thromboangiitis obliterans) or to improvenerve conduction velocity in a patient in need thereof. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical, parenteral,intravenous, intra-arterial, subcutaneous, intramuscular, intracranial,intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, by suppositories,or oral administration. In particular, the inorganic nitrite isadministered in a pharmaceutical composition as described in U.S. patentapplication Ser. No. 12/904,791, hereby incorporated by reference in itsentirety.

Pharmaceutical compositions of inorganic nitrite useful in the methodsof treating pain and microvascular disease or the methods of improvingnerve conduction velocity can contain one or more pharmaceuticallyacceptable carriers. In making a pharmaceutical composition for use inthe methods, the inorganic nitrite, pharmaceutically acceptable salt,solvate, or prodrug thereof is typically mixed with an excipient,diluted by an excipient or enclosed within such a carrier in the formof, for example, a capsule, sachet, paper, or other container. When theexcipient serves as a diluent, it can be a solid, semisolid, or liquidmaterial (e.g., normal saline), which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, and soft and hard gelatincapsules. As is known in the art, the type of diluent can vary dependingupon the intended route of administration. The resulting compositionscan include additional agents, such as preservatives.

The therapeutic agents featuring inorganic nitrite (e.g., sodiumnitrite) can be administered alone, or in a mixture, in the presence ofa pharmaceutically acceptable excipient or carrier. The excipient orcarrier is selected on the basis of the mode and route ofadministration. Suitable pharmaceutical carriers, as well aspharmaceutical necessities for use in pharmaceutical formulations, aredescribed in Remington: The Science and Practice of Pharmacy, 22^(nd)Ed., Allen (2012), a well-known reference text in this field, and in theUSP/NF (United States Pharmacopeia and the National Formulary). Examplesof suitable excipients are lactose, dextrose, sucrose, sorbitol,mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Otherexemplary excipients are described in Handbook of PharmaceuticalExcipients, 6^(th) Edition, Rowe et al., Eds., Pharmaceutical Press(2009).

The compositions useful in the methods can be formulated in a unitdosage form, each dosage containing, e.g., 5 to 100 mg of inorganicnitrite (e.g., sodium nitrite). For example, the dosages can containabout 10 mg to about 50 mg, from about 15 mg to about 40 mg, from about5 mg to about 20 mg, from about 5 mg to about 10 mg, from about 10 mg toabout 20 mg, from about 5 mg to about 15 mg, from about 20 mg to about40 mg, from about 20 mg to about 50 mg; from about 30 mg to about 40 mg,from about 30 mg to about 50 mg, from about 10 mg to about 30 mg, fromabout 70 mg to about 100 mg, from about 60 mg to about 80 mg, from about80 mg to about 100 mg, from about 60 mg to about 90 mg, or from about 70mg to about 85 mg of inorganic nitrite (e.g., sodium nitrite). Forpreparing solid compositions, such as tablets, the inorganic nitrite ismixed with one or more pharmaceutical excipients to form a solid bulkformulation composition containing a homogeneous mixture of a compoundof the present invention. When referring to these bulk formulationcompositions as homogeneous, the inorganic nitrite is typicallydispersed evenly throughout the composition so that the composition canbe readily subdivided into equally effective unit dosage forms such astablets and capsules. This solid bulk formulation is then subdividedinto unit dosage forms of the type described above.

Coatings

The pharmaceutical compositions of inorganic nitrite (e.g., NaNO₂)useful in the methods can be formulated for oral delivery. For instance,tablets or capsules of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of delayedor extended release. The coating may be adapted to release the inorganicnitrite in a predetermined pattern (e.g., in order to achieve acontrolled release formulation). Alternatively, the coating may not beadapted to release the inorganic nitrite or a pharmaceuticallyacceptable salt thereof until after passage of the stomach, e.g., by useof an enteric coating (e.g., polymers that are pH-sensitive (“pHcontrolled release”), polymers with a slow or pH-dependent rate ofswelling, dissolution or erosion (“time-controlled release”), polymersthat are degraded by enzymes (“enzyme-controlled release” or“biodegradable release”) and polymers that form firm layers that aredestroyed by an increase in pressure (“pressure-controlled release”)).

Exemplary enteric coatings that can be used in the pharmaceuticalcompositions including inorganic nitrite include sugar coatings, filmcoatings (e.g., based on hydroxypropyl methylcellulose, methylcellulose,methyl hydroxyethylcellulose, hydroxypropylcellulose,carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/orpolyvinylpyrrolidone), or coatings based on methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and/or ethylcellulose. Furthermore, a time delaymaterial such as, for example, glyceryl monostearate or glyceryldistearate, may be employed.

For example, the tablet or capsule of inorganic nitrite can comprise aninner dosage and an outer dosage component, the latter being in the formof an envelope over the former. The two components can be separated byan enteric layer which serves to resist disintegration in the stomachand permit the inner component to pass intact into the duodenum or to bedelayed in release. When an enteric coating is used, desirably, asubstantial amount of the drug is released in the lower gastrointestinaltract. In addition to coatings that effect delayed or extended release,the solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes (e.g., chemicaldegradation prior to the release of the active drug substance). Thecoating may be applied on the solid dosage form in a similar manner asthat described in Encyclopedia of Pharmaceutical Technology, vols. 5 and6, Eds. Swarbrick and Boyland, 2000.

Formulations for Colonic Drug Release

Colon-targeted drug delivery systems can be used in the methodsfeaturing inorganic nitrite (e.g., NaNO₂). Exemplary approaches include,but are not limited to:

-   -   (a) covalent linkage of the drug with the carrier to form a        prodrug that is stable in the stomach and small intestine and        releases the drug in the large intestine upon enzymatic        transformation by the intestinal microflora; examples of these        prodrugs include azo-conjugates, cyclodextrin-conjugates,        glycoside-conjugates, glucuronate conjugates,        dextran-conjugates, polypeptide and polymeric conjugates;    -   (b) approaches to deliver intact molecule to the colon, such as        coating with pH-sensitive polymers to release the drug at        neutral to alkaline pH, or coating with biodegradable polymers        which release the drug upon degradation by the bacteria in the        colon;    -   (c) embedding the drug in biodegradable matrices and hydrogels        which release the drug in response to the pH or biodegradation;    -   (d) time released systems where once the multicoated formulation        passes the stomach, the drug is released after a lag time of 3-5        hrs which is equivalent to the transit time of the small        intestine;    -   (e) using redox-sensitive polymers where a combination of azo        and disulfide polymers, provide drug release in response to the        redox potential of the colon;    -   (f) using bioadhesive polymers which selectively adhere to the        colonic mucosa slowly releasing the drug; and    -   (g) osmotic controlled drug delivery where the drug is released        through semi-permeable membrane due to osmotic pressure.

Routes of Administration

Inorganic nitrite (e.g., NaNO₂) may be administered to a patient, suchas a patient with pain and/or a microvascular disease or a patient inneed of improved nerve conduction velocity, in a variety of formsdepending on the selected route of administration, as will be understoodby those skilled in the art and as relating to the particular disease orcondition to be treated. The compositions used in the methods ofreducing pain and/or treating a microvascular disease and in the methodsof improving nerve conduction velocity may be administered, for example,by topical, enteral, or parenteral applications. Topical applicationsinclude but are not limited to epicutaneous, inhalation, enema, eyedrops, ear drops, and applications through mucous membranes in the body.Enteral applications include oral administration, rectal administration,vaginal administration, and gastric feeding tubes. Parenteraladministration includes intravenous, intraarterial, intracapsular,intraorbital, intracardiac, intradermal, transtracheal, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural,intrastemal, intraperitoneal, subcutaneous, intramuscular,transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topicalmodes of administration. Parenteral administration may be by continuousinfusion over a selected period of time.

For intravenous or intrathecal delivery or direct injection, thecomposition including inorganic nitrite must be sterile and fluid to theextent that the composition is deliverable by syringe. In addition towater, the carrier can be an isotonic buffered saline solution, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof.Proper fluidity can be maintained, for example, by use of coating, suchas lecithin, by maintenance of required particle size in the case ofdispersion, and by use of surfactants. In many cases, it is preferableto include isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition. Long-termabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

The choice of the route of administration will depend on whether a localor systemic effect is to be achieved using the composition includinginorganic nitrite. For local effects, the composition can be formulatedfor topical administration and applied directly where its action isdesired. For systemic, long term effects, the composition can beformulated for enteral administration and given via the digestive tract.For systemic, immediate, and/or short term effects, the composition canbe formulated for parenteral administration and given by routes otherthan through the digestive tract.

Parenteral Administration

Parenteral depot systems composed of biodegradable polymers are alsouseful in the methods featuring inorganic nitrite. These systems areinjected or implanted into the muscle or subcutaneous tissue to releasethe inorganic nitrite over extended periods of time, ranging fromseveral days to several months. Both the characteristics of the polymerand the structure of the device can control the release kinetics whichcan be either continuous or pulsatile. Polymer-based parenteral depotsystems can be classified as implants or microparticles. The former arecylindrical devices injected into the subcutaneous tissue whereas thelatter are defined as spherical particles in the range of 10-100 μm.Extrusion, compression or injection molding are used to manufactureimplants whereas for microparticles, the phase separation method, thespray-drying technique and the water-in-oil-in-water emulsion techniquesare frequently employed. The most commonly used biodegradable polymersto form microparticles are polyesters from lactic and/or glycolic acid,e.g. poly(glycolic acid) and poly(L-lactic acid) (PLG/PLA microspheres).Of particular interest are in situ forming depot systems, such asthermoplastic pastes and gelling systems formed by solidification, bycooling, or due to the sol-gel transition, cross-linking systems andorganogels formed by amphiphilic lipids. Examples of thermosensitivepolymers used in the aforementioned systems include,N-isopropylacrylamide, poloxamers (ethylene oxide and propylene oxideblock copolymers, such as poloxamer 188 and 407), poly(N-vinylcaprolactam), poly(siloethylene glycol), polyphosphazenes derivativesand PLGA-PEG-PLGA.

Dosing Regimens

Inorganic nitrite (e.g., sodium nitrite (NaNO₂)) can be administered toa patient suffering from pain (e.g., neuropathic pain, inflammatorypain, nociceptive pain, functional pain, musculo-skeletal pain, orcentral nervous system pain) in an amount sufficient to treat or reducethe symptoms of pain (e.g., discomfort, soreness, tightness, stiffness,fatigue, sleeplessness, weakened immune system, depression, anxiety,stress, irritability, or disability). Inorganic nitrite can also beadministered to a patient with a microvascular disease (e.g., peripheralartery disease, diabetic neuropathy, scleroderma, Raynaud's disease,cerebral autosomal dominant arteriopathy with sub-cortical infarcts andleukoencephalopathy, thrombotic microangiopathy, or thromboangiitisobliterans) in an amount sufficient to treat symptoms of microvasculardisease. For example, inorganic nitrite can be administered to a patienthaving a microvascular disease for a first treatment period at a doseeffective to treat or reduce pain, and then administered to the patientfor a second treatment period at a dose effective to treat one or moresymptoms of the microvascular disease (e.g., decreased nitric oxideavailability, physical function, and/or motor neuron activity).

Inorganic nitrite (e.g., NaNO₂) can be administered to a patient for afirst treatment period (e.g., of at least 6 weeks, 7 weeks, 8 weeks, 9weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or longer) totreat or reduce pain (e.g., neuropathic pain, inflammatory pain,nociceptive pain, functional pain, musculo-skeletal pain, or centralnervous system pain). Exemplary doses of inorganic nitrite administered(such as daily, e.g. twice daily) during the first treatment period canbe from about 5 mg to about 50 mg; about 5 mg to about 20 mg; about 5 mgto about 30 mg; about 5 mg to about 40 mg; about 5 mg to about 45 mg;about 10 mg to about 20 mg; about 10 mg to about 30 mg; about 10 mg toabout 40 mg; about 10 mg to about 50 mg; about 15 mg to about 25 mg;about 15 mg to about 35 mg; about 15 mg to about 45 mg; about 20 mg toabout 30 mg; about 20 mg to about 40 mg; about 20 mg to about 50 mg;about 25 mg to about 35 mg; about 25 mg to about 45 mg; about 25 mg toabout 50 mg; about 30 mg to about 40 mg; about 30 mg to about 45 mg;about 30 mg to about 50 mg; about 35 mg to about 40 mg; about 35 mg toabout 45 mg; about 35 mg to about 50 mg; about 40 mg to about 45 mg; orabout 40 mg to about 50 mg. For instance, the amount of inorganicnitrite administered (e.g., twice daily) can be 5 mg, 10 mg, 15 mg, 20mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg during the firsttreatment period.

Following the first treatment period, the amount of inorganic nitrite(e.g., NaNO₂) administered to the patient can be increased during asecond treatment period (e.g., of at least 6 weeks, at least 7 weeks, atleast 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks,at least 12 weeks, at least 13 weeks, at least 14 weeks, or longer) totreat a microvascular disease (e.g., peripheral artery disease, diabeticneuropathy, scleroderma, Raynaud's disease, cerebral autosomal dominantarteriopathy with sub-cortical infarcts and leukoencephalopathy,thrombotic microangiopathy, or thromboangiitis obliterans). Exemplarydoses of inorganic nitrite administered (such as daily, e.g. twicedaily) during the second treatment period can be from about 60 mg toabout 100 mg; about 60 mg to about 70 mg; about 60 mg to about 80 mg;about 60 mg to about 90 mg; about 65 mg to about 75 mg; about 65 mg toabout 85 mg; about 65 mg to about 95 mg; about 70 mg to about 80 mg;about 70 mg to about 90 mg; about 70 mg to about 100 mg; about 75 mg toabout 85 mg; about 75 mg to about 90 mg; about 75 mg to about 100 mg;about 80 mg to about 90 mg; about 80 mg to about 95 mg; about 80 mg toabout 100 mg; about 90 mg to about 100 mg; or about 95 mg to about 100mg of inorganic nitrite. For instance, the amount of inorganic nitriteadministered (e.g., twice daily) can be, e.g., 60 mg, 65 mg, 70 mg, 75mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg during the second treatmentperiod.

The methods also include administering inorganic nitrite (e.g., NaNO₂)for a first treatment period and then a second treatment period at adosage ratio to a patient, such as a patient with pain and/or amicrovascular disease, in which the dosage administered during the firsttreatment period (e.g., to treat pain) is lower than the dosageadministered during the second treatment period (e.g., to treat amicrovascular disease). For example, the ratio of inorganic nitriteadministered during the first treatment period to inorganic nitriteadministered during the second treatment period is approximately 1:20,1:18, 1:16, 1:14, 1:12, 1:10, 1:9, 1:8, 1:7, 3:20, 1:6, 3:16, 1:5, 3:14,2:9, 1:4, 5:18, 2:7, 3:10, 5:16, 1:3, 7:20, 5:14, 3:8, 7:18, 2:5, 5:12,3:7, 7:16, 4:9, 9:20, 1:2, 5:9, 9:16, 4:7, 7:12, 5:8, 9:14, 2:3, 5:7,3:4, or 5:6.

Additionally, the methods can include administering an amount ofinorganic nitrite (e.g., NaNO₂) for a first treatment period (e.g., totreat pain) that is a fractional percentage of the amount of inorganicnitrite administered during the second treatment period (e.g., to treata microvascular disease) to a patient, such as a patient with amicrovascular disease. For instance, the amount of inorganic nitriteadministered during the first treatment period is approximately 5%, 6%,7%, 8%, 10%, 11%, 13%, 14%, 15%, 17%, 19%, 20%, 21%, 22%, 25%, 28%, 29%,30%, 31%, 33%, 35%, 36%, 38%, 39%, 40%, 42%, 43%, 44%, 45%, 50%, 56%,57%, 58%, 63%, 64%, 67%, 71%, 75%, or 83% of the amount of inorganicnitrite administered during the second treatment period.

The amount of inorganic nitrite administered to the patient, such as apatient with pain and/or a microvascular disease, per dose can vary. Forexample, a subject can receive from about 10 μg/kg to about 2,000 μg/kgof inorganic nitrite (e.g., sodium nitrite). Exemplary dosage amounts ofinorganic nitrite include about 20 to about 1000 μg/kg; about 50 toabout 2000 μg/kg; about 100 to about 1500 μg/kg; about 50 μg/kg to about500 μg/kg; about 10 μg/kg to about 250 μg/kg; about 100 μg/kg to about1,000 μg/kg; about 500 μg/kg to about 1500 μg/kg; about 60 to about 1250μg/kg; about 340 to about 750 μg/kg; or about 750 to about 1300 μg/kg.Exemplary dosages of inorganic nitrite can include about 16.5 μg/kg,about 20 μg/kg, about 30 μg/kg, about 50 μg/kg, about 62.5 μg/kg, about100 μg/kg, about 165 μg/kg, about 200 μg/kg, about 500 μg/kg, about 625μg/kg, about 750 μg/kg, about 1000 μg/kg, about 1250 μg/kg, about 1500μg/kg, about 1750 μg/kg, or about 2000 μg/kg. Typically, the amount ofinorganic nitrite contained within a single dose will be an amount thatis effective to treat a condition (e.g., pain and/or a microvasculardisease) as described herein without inducing significant toxicity.

Inorganic nitrite (such as sodium nitrite) can be provided in either asingle or multiple dosage regimens. Dosages of inorganic nitrite (e.g.,sodium nitrite) can be administered, e.g., hourly, bihourly, daily,twice daily, twice a week, three times a week, four times a week, fivetimes a week, six times a week, weekly, biweekly, monthly, bimonthly, oryearly. Additionally, dosages can be administered, e.g., twice, threetimes, four times, five times, six times, seven times, eight times, ninetimes, 10 times, 11 times, or 12 times per day. In particular, thedosing regimen is twice daily. The duration of the dosing regimen canbe, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 day(s), week(s), ormonth(s), or even for the remaining lifespan of the patient with painand/or microvascular disease.

The dosage of inorganic nitrite (e.g., sodium nitrite) administered to apatient with pain, such as chronic pain, can be adjusted based onwhether the patient exhibits a reduction in pain after treatment withthe inorganic nitrite. For instance, a patient with a microvasculardisease (e.g., peripheral artery disease, diabetic neuropathy,scleroderma, Raynaud's disease, cerebral autosomal dominant arteriopathywith sub-cortical infarcts and leukoencephalopathy, thromboticmicroangiopathy, or thromboangiitis obliterans) can exhibit a reductionin pain after administration of inorganic nitrite at a lower dose (e.g.,about 5 mg to about 50 mg twice daily) for a first treatment period(e.g., at least 6 weeks, least 7 weeks, at least 8 weeks, at least 9weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least13 weeks, at least 14 weeks, or longer). The reduction in pain caninclude a decrease in pain intensity, frequency, duration, and/orimprovements in quality of life, which can be determined by, e.g.,performing a Brief Pain Inventory, a Neuropathic Pain Symptom Inventory,and/or a McGill Pain Questionnaire.

When the patient with a microvascular disease exhibits a reduction inpain, the dose of inorganic nitrite (e.g., sodium nitrite) can then beincreased to treat symptoms of microvascular disease, such as decreasednitric oxide availability, physical function, and/or motor neuronactivity. In particular, about 5 mg to about 50 mg of inorganic nitritecan be administered twice daily for a first treatment period (e.g., atleast 6 weeks, least 7 weeks, at least 8 weeks, at least 9 weeks, atleast 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks,at least 14 weeks, or longer) until the patient exhibits a reduction inpain, then about 60 mg to about 100 mg of inorganic nitrite can beadministered twice daily for a second treatment period to treat one ormore symptoms of microvascular disease. Alternatively, whenadministration of inorganic nitrite (e.g., about 5 mg to about 50 mg ofinorganic nitrite twice daily) does not result in a reduction in pain,the first treatment period can be extended (e.g., for 1 week, 2 weeks, 3weeks 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or more) or the doseand/or frequency of inorganic nitrite administration can be changed inorder to determine the amount of inorganic nitrite that results in areduction in pain.

For a patient with a microvascular disease (e.g., peripheral arterydisease, diabetic neuropathy, scleroderma, Raynaud's disease, cerebralautosomal dominant arteriopathy with sub-cortical infarcts andleukoencephalopathy, thrombotic microangiopathy, or thromboangiitisobliterans), the dosage of inorganic nitrite (e.g., sodium nitrite)administered during the second treatment period (e.g., about 60 mg toabout 100 mg twice daily, such as 80 mg twice daily) can also beadjusted based on whether the patient exhibits an improvement in one ormore symptoms of microvascular disease (e.g., decreased nitric oxideavailability, physical function, and/or motor neuron activity relativeto a healthy subject). When administration of inorganic nitrite (e.g.,about 60 mg to about 100 mg twice daily, such as 80 mg twice daily) doesnot result in an improvement in one or more symptoms of microvasculardisease, the second treatment period can be extended (e.g., for 1 week,2 weeks, 3 weeks 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or more)or the dose and/or frequency of inorganic nitrite administration can bechanged in order to determine the amount of inorganic nitrite thatresults in an improvement in one or more symptoms of a microvasculardisease.

Additionally, inorganic nitrite (e.g., sodium nitrite (NaNO₂)) can beadministered to a patient suffering from reduced nerve conductionvelocity (e.g, a patient identified as being in need of improved nerveconduction velocity) to improve nerve conduction velocity in thepatient. For example, a patient that can be administered inorganicnitrite to improve nerve conduction velocity can have a microvasculardisorder, such as diabetic neuropathy, peripheral artery disease,scleroderma, Raynaud's disease, cerebral autosomal dominant arteriopathywith sub-cortical infarcts and leukoencephalopathy, thromboticmicroangiopathy, and thromboangiitis obliterans. In particular, thepatient can be a patient with diabetic neuropathy or peripheral arterydisease. Thus, inorganic nitrite can be administered to a patientidentified as being in need of improved nerve conduction velocity at adosage that increases the nerve conduction velocity of the patient.

For example, inorganic nitrite (e.g., sodium nitrite (NaNO₂)) can beadministered at a dosage of about 60 mg to about 100 mg twice daily(e.g., about 80 mg twice daily) to a patient identified as being in needof improved nerve conduction velocity (e.g., a patient with amicrovascular disease, such as diabetic neuropathy or peripheral arterydisease) for a treatment period sufficient to improve nerve conductionvelocity in the patient. The treatment period can be, e.g., at least 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13weeks, 14 weeks, 15 weeks, or 16 weeks or longer. Exemplary doses ofinorganic nitrite administered daily (e.g., twice daily) to improvenerve conduction velocity can be from about 60 mg to about 100 mg; about60 mg to about 70 mg; about 60 mg to about 80 mg; about 60 mg to about90 mg; about 65 mg to about 75 mg; about 65 mg to about 85 mg; about 65mg to about 95 mg; about 70 mg to about 80 mg; about 70 mg to about 90mg; about 70 mg to about 100 mg; about 75 mg to about 85 mg; about 75 mgto about 90 mg; about 75 mg to about 100 mg; about 80 mg to about 90 mg;about 80 mg to about 95 mg; about 80 mg to about 100 mg; about 90 mg toabout 100 mg; or about 95 mg to about 100 mg of inorganic nitrite (e.g.,sodium nitrite (NaNO₂)). For instance, the amount of inorganic nitriteadministered daily (e.g., twice daily) can be, e.g., 60 mg, 65 mg, 70mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg to improve nerveconduction velocity in a patient in need thereof (e.g., a patient with amicrovascular disease, such as diabetic neuropathy or peripheral arterydisease).

The dosage of inorganic nitrite (e.g., sodium nitrite (NaNO₂))administered to a patient in need of improved nerve conduction velocity,such as patient with a microvascular disease (e.g., diabeticneuropathy), can be adjusted based on whether the patient exhibits animprovement in nerve conduction velocity (e.g., an increase in nerveconduction velocity relative to prior to administration of the sodiumnitrite). For example, the dosage can be increased and/or the treatmentperiod may be extended by, e.g., at least 6 weeks, 7 weeks, 8 weeks, 9weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, or 16weeks or longer, until the patient exhibits an improvement in nerveconduction velocity. Improvements in nerve conduction velocity can bedetermined over the treatment period by, e.g., performing a nerveconduction velocity test. In particular, a nerve conduction velocitytest may be performed by, e.g., attaching two electrode's to a patient'sskin over a test nerve, administering an electrical impulse through oneelectrode to stimulate the nerve, recording the electrical impulsethrough the nerve at the second electrode, and determining the timedifference between stimulation from the first to second electrode (e.g.,in m/s).

After administration of inorganic nitrite (e.g., about 60 mg to about100 mg of inorganic nitrite twice daily, such as 70 mg or 80 mg ofinorganic nitrite twice daily) to the patient, the patient can exhibitan increase in nerve conduction velocity of, e.g, about 1 m/s, about 1.5m/s, about 2 m/s, about 2.5 m/s, about 3 m/s, about 3.5 m/s, about 4m/s, about 4.5 m/s, about 5 m/s, about 5.5 m/s, about 6 m/s, or about6.5 m/s or more relative to the nerve conduction velocity of the patientprior to administration of the inorganic nitrite. A sensory test may beperformed to assess improvements in nerve conduction velocity, such asby placing a monofilament on the patient's skin (e.g., on the patient'sfeet) to assess the patient's ability to sense the monofilament. Forexample, the patient exhibits an increased ability to sense themonofilament after administration of the inorganic nitrite (e.g., about60 mg to about 100 mg of inorganic nitrite twice daily, such as about 80mg twice daily) relative to the ability of the patient to sense themonofilament prior to administration of the inorganic nitrite.

Methods of Treatment

Provided herein are methods for treating or reducing pain in a patient(e.g., a human) and for treating a patient with a microvascular disease,(e.g., peripheral artery disease, diabetic neuropathy, scleroderma,Raynaud's disease, Cerebral Autosomal Dominant Arteriopathy withSub-cortical Infarcts and Leukoencephalopathy, thromboticmicroangiopathy, and Thromboangiitis Obliterans). For instance, patientsexperiencing pain can be treated by the methods, such as byadministering about 5 mg to about 50 mg of inorganic nitrite to thepatient two times per day for, e.g., 6 weeks to 14 weeks, to reducepain. A patient with a microvascular disease can also be treated withthe methods, such as by administering about 5 mg to about 50 mg ofinorganic nitrite to the patient two times per day for, e.g., 6 weeks to14 weeks, to reduce pain, followed by administering about 60 mg to about100 mg of inorganic nitrite (e.g., about 80 mg twice daily) to thepatient two times per day for, e.g., 6 weeks to 14 weeks, to treatsymptoms of microvascular disease. The invention also includes methodsfor improving nerve conduction velocity in a patient (e.g., a human),particularly a patient identified as being in need of improved nerveconduction velocity, such as a patient having a microvascular disease(e.g., diabetic neuropathy, peripheral artery disease, scleroderma,Raynaud's disease, cerebral autosomal dominant arteriopathy withsub-cortical infarcts and leukoencephalopathy, thromboticmicroangiopathy, or thromboangiitis obliterans).

Pain

Pain is associated with a wide range of medical conditions. The presentinvention features inorganic nitrite for use in treating a patient(e.g., a human) with pain or conditions associated with pain. Themethods of treatment are based, inter alia, on the inventor's discoverythat inorganic nitrite (e.g., sodium nitrite) can be administered totreat patients with various forms of pain. Subjects treated using themethods can include subjects with acute pain, subacute pain, or chronicpain (e.g., pain that lasts longer than three to six months or pain thatextends beyond the expected period of healing); or conditions associatedwith pain (e.g., post-herpetic neuralgia, trigeminal neuralgia, phantomlimb pain, carpal tunnel syndrome, sciatica, pudendal neuralgia, complexregional pain syndrome, or central pain syndrome, headaches, inparticular, migraine, joint pain, backaches, sinus pain, muscle pain,nerve pain, and pain affecting specific parts of the body, such asshoulders, pelvis, and neck, and/or pain that is associated with lowerback pain, arthritis, headache, fibromyalgia, shingles, or nervedamage).

Inorganic nitrite (e.g., sodium nitrite) is useful for the treatment orreduction of various forms of pain, whether acute or chronic. Exemplaryconditions that may be associated with pain include, for example, softtissue, joint, and bone inflammation and/or damage (e.g., acute trauma,osteoarthritis, or rheumatoid arthritis), myofascial pain syndromes(fibromyalgia), headaches (including cluster headache, migraine, andtension type headache), myocardial infarction, angina, ischemiccardiovascular disease, post-stroke pain, sickle cell anemia, peripheralvascular occlusive disease, cancer, inflammatory conditions of the skinor joints, diabetic neuropathy, and acute tissue damage from surgery ortraumatic injury (e.g., burns, lacerations, or fractures).

For example, the present invention provides methods of administeringinorganic nitrite to alleviate neuropathic pain. Neuropathic pain cantake a variety of forms depending on its origin and can be characterizedas acute, subacute, or chronic depending on the duration. The pain maybe described as being peripheral neuropathic if the initiating injuryoccurs as a result of a complete or partial transection of a nerve ortrauma to a nerve plexus. Peripheral neuropathy can result fromtraumatic injuries, infections, metabolic disorders, diabetes, and/orexposure to toxins. Alternatively, neuropathic pain is described asbeing central neuropathic following a lesion to the central nervoussystem, such as a spinal cord injury or a cerebrovascular accident.Types of neuropathic pain that can be treated with inorganic nitrite(e.g., sodium nitrite) include, but are not limited to, diabeticperipheral neuropathy, post-herpetic neuralgia, trigeminal neuralgia,phantom limb pain, carpal tunnel syndrome, sciatica, pudendal neuralgia,complex regional pain syndrome, sensory polyneuropathies,mono-neuropathies, and central pain syndrome.

Additionally, methods for treating inflammatory pain by administeringinorganic nitrite (e.g., sodium nitrite) are provided. Inflammatory painis a form of pain caused by tissue injury or inflammation (e.g., inpostoperative pain or rheumatoid arthritis). Following a peripheralnerve injury, symptoms are typically experienced in a chronic fashion,distal to the site of injury and are characterized by hyperesthesia(enhanced sensitivity to a natural stimulus), hyperalgesia (abnormalsensitivity to a noxious stimulus), allodynia (widespread tendernessassociated with hypersensitivity to normally innocuous tactile stimuli),and/or spontaneous burning or shooting lancinating pain. In inflammatorypain, symptoms are apparent, at least initially, at the site of injuryor inflamed tissues and typically accompany arthritis-associated pain,musculo-skeletal pain, and postoperative pain. The different types ofpain may coexist or pain may be transformed from inflammatory toneuropathic during the natural course of the disease, as inpost-herpetic neuralgia.

Inorganic nitrite (e.g., sodium nitrite) can also be used for thetreatment, reduction, or prevention of musculo-skeletal pain (aftertrauma, infections, and exercise), pain caused by spinal cord injury,tumors, compression, inflammation, dental pain, episiotomy pain, deepand visceral pain (e.g., heart pain, bladder pain, or pelvic organpain), muscle pain, eye pain, orofacial pain (e.g., odontalgia,trigeminal neuralgia, glossopharyngeal neuralgia), abdominal pain,gynecological pain (e.g., dysmenorrhea and labor pain), pain associatedwith nerve and root damage due to trauma, compression, inflammation,toxic chemicals, hereditary conditions, central nervous system pain,such as pain due to spinal cord or brain stem damage, cerebrovascularaccidents, tumors, infections, demyelinating diseases including multiplesclerosis, low back pain, sciatica, and post-operative pain.

Microvascular Diseases

The present invention features the use of inorganic nitrite to treat asubject (e.g., a human) with a microvascular disease. For instance,inorganic nitrite (e.g., sodium nitrite) can be administered to treatpatients with peripheral artery disease. In particular, patients withperipheral artery disease can be treated by administering about 5 mg toabout 50 mg of inorganic nitrite (e.g., 40 mg of sodium nitrite) to thepatient two times per day for 6 weeks to 14 weeks; and thenadministering about 60 mg to about 100 mg of inorganic nitrite (e.g., 80mg of sodium nitrite) to the patient two times per day for 6 weeks to 14weeks. The methods are useful for treating symptoms of peripheral arterydisease, such as intermittent claudication, often described by patientsas a cramping, aching, or fatigue sensation in the calf muscles of thelegs that occurs during physical activity; limited exercise tolerance;coldness or changes in skin color of the lower extremities; hair loss orslower hair growth on your feet and legs; and a weak pulse in your feetand legs.

Likewise, inorganic nitrite (e.g., sodium nitrite) can be administeredto treat patients having diabetic neuropathy. For instance, patientswith diabetic neuropathy can be treated by administering about 5 mg toabout 50 mg of inorganic nitrite (e.g., 40 mg of sodium nitrite) to thepatient two times per day for 6 weeks to 14 weeks; and thenadministering about 60 mg to about 100 mg of inorganic nitrite (e.g., 80mg of sodium nitrite) to the patient two times per day for 6 weeks to 14weeks. In particular, the methods are useful for treating symptoms ofdiabetic neuropathy including, but not limited to, neuropathic pain,nerve damage, numbness, loss of balance, foot deformities, burningsensations, or tingling.

Moreover, inorganic nitrite (e.g., sodium nitrite) can be administeredto treat patients with scleroderma. For example, patients withscleroderma can be treated by administering about 5 mg to about 50 mg ofinorganic nitrite (e.g., 40 mg of sodium nitrite) to the patient twotimes per day for 6 weeks to 14 weeks; and then administering about 60mg to about 100 mg of inorganic nitrite (e.g., 80 mg of sodium nitrite)to the patient two times per day for 6 weeks to 14 weeks. Thus, themethods are useful for treating symptoms of scleroderma including, butnot limited to, hardening and tightening of patches of skin; numbness,pain, or color changes in the fingers or toes; acid reflux and digestiveproblems; ulcers or sores on fingertips; small red spots on the face andchest; opened blood vessels; puffy, painful, or swollen joints; muscleweakness; dry eyes or mouth; swelling of the hands and fingers;shortness of breath; and weight loss.

Inorganic nitrite (e.g., sodium nitrite) can also be administered, asdescribed herein, to treat patients with Raynaud's syndrome. Inparticular, patients with Raynaud's syndrome can be treated byadministering about 5 mg to about 50 mg of inorganic nitrite (e.g., 40mg of sodium nitrite) to the patient two times per day for 6 weeks to 14weeks; and then administering about 60 mg to about 100 mg of inorganicnitrite (e.g., 80 mg of sodium nitrite) to the patient two times per dayfor 6 weeks to 14 weeks. Accordingly, the methods are useful fortreating symptoms of Raynaud's syndrome including, but not limited to,cold fingers or toes, color changes in your skin in response to cold orstress, numbness, or pain in the extremities.

For instance, inorganic nitrite can also be administered to treatpatients with Cerebral Autosomal Dominant Arteriopathy with Sub-corticalInfarcts and Leukoencephalopathy (CADASIL). In particular, patients withCADASIL can be treated by administering about 5 mg to about 50 mg ofinorganic nitrite (e.g., 40 mg of sodium nitrite) to the patient twotimes per day for 6 weeks to 14 weeks; and then administering about 60mg to about 100 mg of inorganic nitrite (e.g., 80 mg of sodium nitrite)to the patient two times per day for 6 weeks to 14 weeks. Thus, themethods are useful for alleviating symptoms of CADASIL including, butnot limited to, transient ischemic attacks, cerebral infarction,dementia, psychiatric disturbances, recurrent strokes, and migraine withaura.

Additionally, inorganic nitrite can be administered to treat patientswith thrombotic microangiopathy. For example, patients with thromboticmicroangiopathy can be treated by administering about 5 mg to about 50mg of inorganic nitrite (e.g., 40 mg of sodium nitrite) to the patienttwo times per day for 6 weeks to 14 weeks; and then administering about60 mg to about 100 mg of inorganic nitrite (e.g., 80 mg of sodiumnitrite) to the patient two times per day for 6 weeks to 14 weeks. Inparticular, the methods are useful for treating symptoms of thromboticmicroangiopathy including, but not limited to, fatigue, dizziness,shortness of breath, bruises, fever, microangiopathic hemolytic anemia,renal failure or complications, thrombocytopenia, neurologicalmanifestations, and seizures.

Inorganic nitrite (e.g., sodium nitrite) can also be administered, asdescribed herein, to treat patients with Thromboangiitis Obliterans. Inparticular, patients with Thromboangiitis Obliterans can be treated byadministering about 5 mg to about 50 mg of inorganic nitrite (e.g., 40mg of sodium nitrite) to the patient two times per day for 6 weeks to 14weeks; and then administering about 60 mg to about 100 mg of inorganicnitrite (e.g., 80 mg of sodium nitrite) to the patient two times per dayfor 6 weeks to 14 weeks. Accordingly, the methods are useful fortreating symptoms of Thromboangiitis Obliterans including, but notlimited to, color changes in the fingers or toes; cold fingers or toes;pain in the hands and feet, particularly in exposure to cold or withemotional stress; intermittent claudication; or small painful ulcers onfingers or toes.

Nerve Conduction Velocity

The present invention features inorganic nitrite for use in treating apatient (e.g., a human) identified as being in need of improved nerveconduction velocity. The methods of treatment are based, inter alia, onthe inventor's discovery that inorganic nitrite (e.g., sodium nitrite)can be administered to treat a patient in need of improved nerveconduction velocity, such as a patient identified as having reducednerve conduction velocity relative to a healthy subject (e.g., a subjecthaving a nerve conduction velocity of greater than about 45 m/s, such asabout 50 m/s to about 60 m/s). For example, a patient identified asbeing in need of improved nerve conduction velocity may have a nerveconduction velocity of, e.g., less than 45 m/s, such as about 35 m/s,about 36 m/s, about 37 m/s, about 38 m/s, about 39 m/s, about 40 m/s,about 41 m/s, about 42 m/s, about 43 m/s, or about 44 m/s.

A patient (e.g., a patient having a microvascular disease, such asdiabetic neuropathy, peripheral artery disease, scleroderma, Raynaud'sdisease, cerebral autosomal dominant arteriopathy with sub-corticalinfarcts and leukoencephalopathy, thrombotic microangiopathy, orthromboangiitis obliterans) may be identified as being in need ofimproved nerve conduction velocity using a monofilament, such as in amonofilament test. For example, the bent monofilament (e.g., exerting apressure of 10 grams) or unbent monofilament may be touched to differentpoints on the sole of the patient's foot, and the patient will identifyif they sense the monofilament. If the patient is unable to sense a bentmonofilament, e.g., exerting 10 grams of pressure on the patient's foot,then the patient is identified as being in need of improved nerveconduction velocity. Inorganic nitrite (e.g., sodium nitrite) can thenbe administered to improve nerve conduction velocity in the patient. Forexample, administration of about 60 mg to about 100 mg of inorganicnitrite twice daily (e.g., about 70 mg or about 80 mg twice daily) canresult in an improved ability of the patient to sense the monofilament.In particular, the patient exhibits an improved ability to sense themonofilament after administration of inorganic nitrite for a treatmentperiod sufficient to improve nerve conduction velocity in the patient(e.g., a treatment period of at least 6 weeks, at least 7 weeks, atleast 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks,at least 12 weeks, at least 13 weeks, or at least 14 weeks or longer).

Inorganic nitrite (e.g., sodium nitrite) can be administered to improvenerve conduction velocity in a patient (e.g., a human) with amicrovascular disease. For example, administration of about 60 mg toabout 100 mg of inorganic nitrite twice daily (e.g., about 70 mg orabout 80 mg twice daily) can increase nerve conduction velocity in apatient with a microvascular disease, such as diabetic neuropathy,peripheral artery disease, scleroderma, Raynaud's disease, cerebralautosomal dominant arteriopathy with sub-cortical infarcts andleukoencephalopathy, thrombotic microangiopathy, or thromboangiitisobliterans. Thus, inorganic nitrite can be administered to a patientwith any of these microvascular disease to increase the nerve conductionvelocity (e.g., by about 1 m/s, about 1.5 m/s, about 2 m/s, about 2.5m/s, about 3 m/s, about 3.5 m/s, about 4 m/s, about 4.5 m/s, about 5m/s, about 5.5 m/s, about 6 m/s, or about 6.5 m/s or more relative tothe patient prior to administration of the inorganic nitrite) of thepatient. In particular, the patient having a microvascular diseaseexhibits an increase in nerve conduction velocity after administrationof inorganic nitrite for a treatment period sufficient to improve nerveconduction velocity in the patient (e.g., a treatment period of at least6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, or atleast 14 weeks or longer).

Additionally, inorganic nitrite can be administered to a patient thatexhibits deficits in balance and/or a reduced ability to sense painrelative to a healthy subject as a result of reduced nerve conductionvelocity. For example, administration of about 60 mg to about 100 mg ofinorganic nitrite twice daily (e.g., about 70 mg or about 80 mg twicedaily) to a patient in need thereof (e.g., a patient identified ashaving deficits in balance and/or a reduced ability to sense pain) canincrease the nerve conduction velocity (e.g., by about 1 m/s, about 1.5m/s, about 2 m/s, about 2.5 m/s, about 3 m/s, about 3.5 m/s, about 4m/s, about 4.5 m/s, about 5 m/s, about 5.5 m/s, about 6 m/s, or about6.5 m/s or more relative to the patient prior to administration of theinorganic nitrite) of the patient. Moreover, the increase in nerveconduction velocity of the patient can be sustained throughadministration of the inorganic nitrite, e.g., for a treatment period ofat least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks,at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13weeks, or at least 14 weeks or longer. For instance, the nerveconduction velocity increase to greater than 45 m/s and remains atgreater than 45 m/s during treatment with the inorganic nitrite (e.g.,sodium nitrite). Thus, the methods featuring the administration ofinorganic nitrite are useful for improving a patient's ability tobalance and sense pain by increasing the nerve conduction velocity ofthe patient.

Example 1. Clinical Studies of Sodium Nitrite

Sodium nitrite was investigated as a therapy for patients withmicrovascular disease, particularly patients with peripheral arterialdisease (PAD) and patients with diabetic neuropathy. The overall goal ofthis dose-ranging study was to evaluate the efficacy of low and highdoses of oral sodium nitrite (e.g., 40 mg sodium nitrite and 80 mgsodium nitrite, respectively) for treating pain and the pathophysiologyassociated with the microvascular diseases of PAD and diabeticneuropathy.

Two chronic dosing clinical trials were performed to study the safetyand biological activity of an immediate release formulation of sodiumnitrite (TV1001) in patients with PAD and a sustained releaseformulation of sodium nitrite (TV1001sr) in patients with diabeticneuropathy. The first trial included 55 human patients with PAD, inwhich 70% of patients also had diabetes. The PAD patients wererandomized to receive one of the treatment regimens of placebo, 40 mgTV1001 twice daily (BID), or 80 mg TV1001 BID. The second trial included30 patients with diabetic neuropathy. The diabetic neuropathy patientswere randomized to receive one of the treatment regimens of placebo, 40mg TV1001sr BID, or 80 mg TV1001sr BID. The trial with PAD patients wasconducted for 10 weeks, and the trial with diabetic neuropathy patientswas conducted for 12 weeks. Due to a manufacturing problem, theeffective doses of TV1001sr were 35 mg and 70 mg during the trialperiod.

Example 2. Sodium Nitrite Decreases Pain in PAD Patient

Pain in PAD patients was assessed using a standardized pain scale scorein which a higher score indicates less reported pain and a lower scoreindicates greater reported pain. Pain scores for the PAD patients weredetermined at baseline and after 10 weeks of receiving placebo, 40 mgTV1001 BID, or 80 mg TV1001 BID. Patients receiving 40 mg TV1001 BID hada pain scale score of 11.90 at day 70 of treatment, patients receiving80 mg TV1001 BID had a pain scale score of −3.13 at day 70 of treatment,and patients receiving placebo had a pain scale score of −2.12 at day 70of treatment (Table 1). Thus, patients receiving 40 mg TV1001 BIDreported significantly less pain than the placebo group (p<0.05),whereas the 80 mg group reported a similar amount of pain as the placebogroup. These results show that administration of the low dose (40 mg) ofsodium nitrite twice daily resulted in a decrease in pain experienced byPAD patients in comparison to administration of the high dose (80 mg) ofsodium nitrite twice daily or placebo.

TABLE 1 Pain scale scores of PAD patients administered placebo, 40 mgTV1001 BID, or 80 mg TV1001 BID. TV1001 TV1001 TV1001 40 mg 80 mgCombined Placebo (N = 19) (N = 18) (N = 37) (N = 18) P-value Pain ScaleLeast Squares 11.90 −3.13 4.39 −2.12 Score: Visit 6 Mean (Day 70)/EarlyTermination- Visit 1 (Day 0) Std. Error 4.529 4.944 3.300 5.050 TV100140 mg vs. 0.033 TV1001 80 mg TV1001 40 mg vs. 0.046 Placebo TV1001 80 mgvs. 0.887 Placebo TV1001 40 mg and 0.287 80 mg combined vs. Placebo

Example 3. Sodium Nitrite Decreases Pain in Diabetic Neuropathy Patients

Pain in 28 patients with diabetic neuropathy during the clinical trialwas evaluated using the Brief Pain Inventory (BPI), the Neuropathic PainSymptom Inventory (NPSI), and the McGill Pain Index (MPI).

The BPI is a questionnaire in which patients rate the severity of painand degree of interference with function (see Cleeland & Ryan, Ann.Acad. Med. Singapore. 23(2):129-38, 1994; hereby incorporated byreference in its entirety). The different symptoms of neuropathic painwere evaluated using the NPSI (see Bouhassira et al. Pain.108(3):248-57, 2004; hereby incorporated by reference in its entirety).The MPI was used to determine continuous and intermittent patterns ofpain experienced by the patients (see Melzack. Pain. 1 (3):277-99, 1975;hereby incorporated by reference in its entirety). A lower scoresindicates less reported pain and a higher score indicates greaterreported pain for the BPI, NPSI, and MPI.

Diabetic neuropathy subjects receiving 40 mg TV1001sr BID and 80 mgTV1001sr BID reported less pain after 12 weeks of treatment (visit 3(V3)) based on the NPSI and the severity score of the BPI (Table 2). Acomparison of V3 data for the NPSI to V1 data showed that subjectsreceiving 40 mg TV1001sr reported approximately 1.5 fold less pain andsubjects receiving 80 mg TV1001sr reported approximately 2.6 fold lesspain at V3 than subjects in the placebo group. The same trend wasobserved in the Severity Score of the BPI, in which subjects receiving40 mg TV1001sr reported approximately 1.97 fold less pain and subjectsreceiving 80 mg TV1001sr reported approximately 2.34 fold less pain atV3 than subjects in the placebo group. For the MPI, patients receiving40 mg TV1001sr reported slightly less pain than the placebo group, whilepatients receiving 80 mg TV1001sr reported very little reduction in painusing the MPI.

TABLE 2 Changes in Brief Pain Inventory (BPI), Neuropathic Pain SymptomInventory Questionnaire (NPSI), and McGill Pain Index (MPI) scores ofdiabetic neuropathy patients administered placebo, 40 mg TV1001sr BID,or 80 mg TV1001sr BID from baseline (V1) to 12 weeks (V3). % V2 V3Change TV1001sr Fold Change Change of V3 to Improvement Brief PainInventory V1 from V1 from V1 V1 Over Placebo Severity Score Placebo 5.1−0.96 −0.24 5.9% 40 mg 4.3 −1.06 −0.48 11.6% 1.97 80 mg 5.9 −0.54 −0.8413.6% 2.34 Interference Score Placebo 5.0 −0.57 −0.73 14.0% 40 mg 4.4−1.11 −0.20 4.5% −3.11 80 mg 6.4 0.62 0.77 10.94% −1.28 Neuropathic PainSymptom Inventory Total Score Placebo 47.4 −8.44 −4.00 8.4% 40 mg 34.7−4.71 −4.43 12.7% 1.51 80 mg 56.0 −1.88 −10.00 22.0% 2.62 McGill PainIndex Total Score Placebo 5.1 −1.4 −1.5 29.4% 40 mg 3.9 −0.7 −1.4 35.9%1.22 80 mg 4.8 0.8 −0.2 4.2% −7.00 Continuous Pain Placebo 5.0 −1.9 −1.836.0% 40 mg 3.7 −0.2 −1.7 48.6% 1.35 80 mg 4.4 1.2 −0.1 2.3% −15.7Intermittent Pain Placebo 6.0 −1.6 −1.9 31.7% 40 mg 4.6 −1.1 −1.8 39.1%1.23 80 mg 5.9 0.7 −0.6 10.2% −3.11

Although all diabetic neuropathy subjects reported a decrease in pain inthe Total Score section of the NPSI questionnaire, diabetic neuropathysubjects receiving 40 mg TV1001sr BID and 80 mg TV1001sr BID reportedthe greatest reduction in pain at V3 (Table 3). The 40 mg dose groupreported significantly less pain than the 80 mg treatment group atbaseline and throughout the study (ANOVA, df=2, F=4.38, p=0.02).

TABLE 3 Summary of Neuropathic Pain Symptom Inventory Questionnaire(NPSI) scores of diabetic neuropathy patients administered placebo, 40mg TV1001sr BID, or 80 mg TV1001sr BID from baseline (V1) to 12 weeks(V3). Visit 1 Visit 2 Visit 3 NPSI Total Placebo Mean 47.4 39.0 43.4S.D. 25.7 24.4 25.3 N 9 9 9 40 mg TV1001sr Mean 34.7 30.0 30.3 S.D. 22.222.7 26.2 N 7 7 7 80 mg TV1001sr Mean 56.0 54.1 46.0 S.D. 18.0 16.2 23.9N 8 8 8

Pain in diabetic neuropathy subjects receiving 40 mg TV1001sr BID and 80mg TV1001sr BID was also assessed using the Total Severity and TotalInterference scores of the BPI (Table 4). In both of the BPI sections,subjects receiving 40 mg TV1001sr BID reported significantly less painat baseline (V1) and throughout the trial period as did the 80 mg dosegroup (ANOVA, Total Severity: F=3.39, p=0.04; Total Interference:F=4.82, p=0.01). For the Total Severity BPI scores, subjects receiving40 mg TV1001sr BID and 80 mg TV1001sr also exhibited the greatestreported decrease in pain.

TABLE 4 Summary of Brief Pain Inventory (BPI) Total Severity and TotalInterference scores of diabetic neuropathy patients administeredplacebo, 40 mg TV1001sr BID, or 80 mg TV1001sr BID from baseline (V1) to12 weeks (V3). Visit 1 Visit 2 Visit 3 BPI Total Placebo Mean 5.1 4.14.8 Severity S.D. 2.1 2.6 2.3 N 9 9 9 40 mg TV1001sr Mean 4.3 3.2 3.8S.D. 2.6 1.9 2.6 N 7 7 7 80 mg TV1001sr Mean 5.9 5.4 5.1 S.D. 1.9 1.32.1 N 8 8 8 BPI Total Placebo Mean 5.0 4.4 4.3 Interference S.D. 3.4 3.04.0 N 9 9 9 40 mg TV1001sr Mean 4.4 3.2 4.2 S.D. 2.4 2.6 2.6 N 7 7 7 80mg TV1001sr Mean 6.4 7.1 5.7 S.D. 2.3 1.6 2.1 N 8 8 8

Pain in diabetic neuropathy subjects receiving 40 mg TV1001sr BID and 80mg TV1001sr BID was also assessed using the McGill Questionnaire TotalIntermittent pain score, Total Continuous pain score, and Total painscore (Table 5). For the McGill Total score (ANOVA, F=3.67, p=0.03) andthe Intermittent score (ANOVA, F=3.98, p=0.02), the 40 mg TV1001subjects had significantly lower scores than the 80 mg group. Unlike theother pain questionnaires, the subjects treated with 80 mg TV1001srreported very little reduction in pain as assessed with the MPI, wellless than that reported by subjects in the placebo group.

TABLE 5 Summary of McGill Pain Index (MPI) scores of diabetic neuropathypatients administered placebo, 40 mg TV1001sr BID, or 80 mg TV1001sr BIDfrom baseline (V1) to 12 weeks (V3). Visit 1 Visit 2 Visit 3 McGillPlacebo Mean 5.1 3.7 3.6 Total (N = 9) S.D. 2.4 2.0 2.5 40 TV1001sr Mean3.9 3.1 2.5 (N = 7) S.D. 2.4 1.9 2.4 80 TV1001sr Mean 4.8 5.6 4.6 (N =8) S.D. 2.2 2.0 2.1 McGill Placebo Mean 5.0 3.1 3.2 Continuous (N = 9)S.D. 3.0 2.2 2.2 40 TV1001sr Mean 3.7 3.5 1.9 (N = 7) S.D. 2.5 3.5 2.280 TV1001sr Mean 4.4 5.6 4.3 (N = 8) S.D. 2.5 2.5 2.1 McGill PlaceboMean 6.0 4.4 4.1 Intermittent (N = 9) S.D. 2.9 2.7 3.6 40 TV1001sr Mean4.6 3.5 2.8 (N = 7) S.D. 2.5 2.4 2.9 80 TV1001sr Mean 5.9 6.6 5.3 (N =8) S.D. 3.0 2.2 2.4

Diabetic neuropathy subjects receiving 40 mg TV1001sr BID, 80 mgTV1001sr BID, or placebo were also given a log book and instructed torecord the level of pain experienced on each day at the time oftreatment. Subjects in the 40 mg TV1001sr BID group reported a quickerreduction in pain and an overall greater reduction in pain than eitherthe placebo or 80 mg TV1001sr BID groups (FIG. 3). When the means of thefinal visit were subtracted from the baseline value for each treatmentgroup, the 40 mg TV1001sr BID treatment group reported the greatestreduction in pain, a mean drop of 4.0 (SD=4.3), while the placebo groupreported a mean drop of 2.1 (SD=0.7). Very little change in painperception was reported by subjects in the 80 mg TV1001sr BID treatmentgroup, with a mean drop of only 0.5 (SD=1.4).

Example 4. Sodium Nitrite Improves Symptoms of Microvascular Disease

The biological activity of sodium nitrite was assessed by flow-mediateddilation (FMD) for PAD patients at baseline and after 10 weeks ofreceiving placebo, 40 mg TV1001 BID, or 80 mg TV1001 BID. FMD is anoninvasive method to measure endothelial dysfunction using, e.g.,Brachial Artery Ultrasound Imagining (see Peretz et al. BMC Cardiovasc.Disord. 7: 11, 2007; hereby incorporated by reference in its entirety).The Exploratory Group consisted of PAD patients that completed theprotocol as instructed and took >70% of their medication. PAD patientsreceiving 80 mg TV1001 BID exhibited the most significant improvement inFMD for all groups from baseline (FIG. 1). In particular, there was adose dependent improvement in FMD in the Exploratory Group with patientsreceiving 80 mg TV1001 BID exhibiting the greatest increase in FMD.Notably, diabetic PAD patients demonstrated a statistically significantimprovement in FMD.

The RAND-36 Questionnaire was used to assess changes in quality of lifeof PAD patients at baseline and after 10 weeks of receiving placebo, 40mg TV1001 BID, or 80 mg TV1001 BID. The RAND-36 Questionnaire is a36-item, patient-reported survey of patient health. PAD patients wereevaluated in the RAND-36 Questionnaire categories of PhysicalFunctioning, Limitations due to Physical Health, Energy/Fatigue,Improvements in Pain, and General Health (FIG. 2). For the PhysicalFunctioning scale, patients receiving 80 mg TV1001 BID exhibited agreater change from the baseline score after 10 weeks of treatmentrelative to patients receiving 40 mg TV1001 BID or the patientsreceiving placebo. Thus, the 80 mg dose provided the greatest benefit inphysical function of PAD patients.

For the RAND-36 Questionnaire, patients receiving 40 mg TV1001 BID and80 mg TV1001 BID exhibited a similar increase in the change frombaseline for the Energy/Fatigue score relative to patients receiving theplacebo. Accordingly, both 40 mg TV1001 BID and 80 mg TV1001 BIDappeared to improve levels of energy and fatigue in PAD patients.Additionally, patients receiving 40 mg TV1001 BID exhibited a greaterimprovement in pain relative to patients receiving 80 mg TV1001 BID orthe placebo patients. These results corroborate that low dose (40 mg)sodium nitrite decreases pain in diabetic neuropathy patients, as isdescribed in Example 3.

Example 5. Sodium Nitrite Improves Nerve Conduction Velocity in DiabeticNeuropathy Patients

Nerve conduction velocity was also measured as a biological indicator ofnerve health in patients with diabetic neuropathy receiving 40 mgTV1001sr BID, 80 mg TV1001sr BID, or placebo. For Visit 1, Visit 2, andVisit 3, three conduction measures were averaged and three velocitymeasures were averaged. Nerve conduction velocity improved in patientsreceiving 80 mg TV1001sr BID in comparison to patients receiving 40 mgTV1001sr BID or placebo (FIG. 4).

The nerve conduction velocity measures were also analyzed using anAnalysis of Variance with one between-subjects factor (Group: bothplacebo groups combined, 40 mg TV1001sr BID, and 80 mg TV1001sr BID) andone within-subjects factor (Visit: Visit 1, Visit 2, and Visit 3). Therewere significant differences among the groups for the conductionmeasures, but not for the velocity measures. There was no significanteffect for the Visit factor or for the Group by Visit interaction foreither conduction or velocity. There was a significant effect for Group.Specifically, the velocity values for the 80 mg TV1001sr BID group weresignificantly higher than the values for the 40 mg TV1001sr BID group.Thus, administration of the high dose (80 mg) formulation of sodiumnitrite twice daily resulted in a statistically significant improvementin nerve conduction velocity in patients with diabetic neuropathy over atreatment period of ten weeks.

There were no significant differences in Quantitative Sensory Testing atbaseline among the groups, the average sensory conductance was higherfor the 80 mg TV1001sr BID group than the placebo or 40 mg TV1001sr BIDgroup, and average sensory velocity was lower for the 80 mg TV1001sr BIDgroup (Table 6). Nerve sensory conductance showed very little changebetween baseline testing and Visits 1 and 2 for the placebo and 40 mgTV1001sr BID group, but trended toward decreasing for the 80 mg TV1001srBID group (p=0.154). Similarly, nerve sensory velocity remained stablefor the placebo and 40 mg TV1001sr BID group, but exhibited a trendtowards increasing (p=0.116) with continued improvement for the 80 mgTV1001sr BID group.

TABLE 6 Efficacy analysis of Quantitative Sensory Testing (QST) fordiabetic neuropathy patients administered placebo, 40 mg TV1001sr BID,or 80 mg TV1001sr BID. Treatment Group TV1001sr TV1001sr Placebo 40 mg80 mg (n = 9) (n = 6) (n = 8) Nerve Conductance Baseline: Mean (SD)  3.9(1.4)  3.4 (0.7)  6.0 (2.9) Visit 2: Mean (SD)  4.3 (1.8)  3.1 (0.9) 5.2 (3.0) Visit 3: Mean (SD)  3.5 (1.2)  3.2 (0.6)  4.2 (1.5) NerveVelocity Baseline: Mean (SD) 44.7 (6.5) 41.1 (4.3) 39.4 (8.2) Visit 2:Mean (SD) 40.1 (5.3) 39.3 (7.8)  42.3 (10.7) Visit 3: Mean (SD) 43.2(6.4) 37.7 (3.2) 46.8 (4.2)

Example 6: Reduction in Headaches with Sustained Release Formulation ofSodium Nitrite

All subjects in the placebo and 40 mg TV1001sr BID treatment group, and7 of the 9 subjects in the 80 mg TV1001sr BID treatment group, reportedat least one adverse event (AE). The total number of reported AEs wasslightly higher for the placebo group, but not really different fromthose in the TV1001sr groups (Table 7). Importantly, headaches, whichwas the most common AE reported previously for an immediate releaseformulation of sodium nitrite were not reported by any subjects in the40 mg TV1001sr BID treatment group and were reported by the same numberof subjects in the 80 mg TV1001sr BID treatment group as in the placebogroup (2 subjects each).

TABLE 7 Summary of adverse events for diabetic neuropathy patientsadministered placebo, 40 mg TV1001sr BID, or 80 mg TV1001sr BID.Treatment TV1001sr TV1001sr Placebo 40 mg 80 mg (n = 9) (n = 8) (n = 9)Number (%) of 9 (100.0%) 8 (100.0%) 7 (77.8%) Subjects with at least oneAE Number of AEs 29 23 23 Number of SAEs 2 5 2 Numbers of AEs bySeverity Mild 23 14 17 Moderate 2 5 4 Severe 1 4 1 Not Recorded 3 0 1Number of AEs by Relationship to Study Drug Not Related 24 19 19Possibly Related 5 4 4 Probably Related 0 0 0 AEs Appearing Related toPrevious Exposure to Study Drug Dose Headache 2 (22.2%) 0 (0%) 2 (22.2%)Not Related 1 (11.1%) 0 (0%) 0 (0%) Possibly Related 1 (11.1%) 0 (0%) 2(22.2%) Dizziness (incl. 2 (22.2%) 2 (25%) 2 (22.2%) shakiness) NotRelated 0 (0) 1 (12.5%) 1 (11.1%) Possibly Related 2 (22.2%) 1 (12.5%) 1(11.1%)

These results indicate that treatment with TV1001sr appears tolerable inpatients with diabetic neuropathy and the use of the sustained releaseformulation seems to eliminate the headaches and dizziness noted inprior studies of an immediate release formulation. There was a trendacross questionnaires that demonstrated a potential benefit,particularly of 40 mg TV1001sr BID, in reducing total pain at the end ofthe trial period compared to that at baseline. A trend toward improvingnerve function was observed following treatment with 80 mg TV1001 sr.Thus, these results demonstrates that TV1001sr eliminated headaches anddizziness.

Example 7. Summary of Clinical Studies Featuring Sodium NitriteAdministered Twice Daily

Administration of low dose (40 mg) sodium nitrite twice daily resultedin a statistically significant reduction in pain for PAD and diabeticneuropathy patients, while administration of the high dose (80 mg) ofsodium nitrite twice daily improved symptoms of microvascular disease inPAD and diabetic neuropathy patients. Additionally, administration ofthe sustained release formulation of sodium nitrite (TV1001sr) twicedaily eliminated headaches and dizziness associated with TV1001.

Based on these results, an appropriate dosing schedule will be to treata patient (e.g., a patient with PAD or diabetic neuropathy) that haschronic pain due to microvascular disease with about 5 mg to about 50 mgTV1001sr BID (e.g., 40 mg TV1001sr BID) for 6 weeks to 14 weeks (e.g.,10 weeks) to reduce pain. Then, the dosage of TV1001sr would beincreased to about 60 mg to about 100 mg administered BID (e.g., 80 mgTV1001sr BID) to improve and maintain the biological response to sodiumnitrite in patients with microvascular disease.

For diabetic neuropathy patients, Brief Pain Inventory (BPI),Neuropathic Pain Symptom Inventory Questionnaire (NPSI), and McGill PainIndex (MPI) scores in addition to daily pain logs indicated thattreatment with 40 mg TV1001sr BID resulted in a reduction in pain. Painwas also reduced in diabetic neuropathy subjects receiving 80 mgTV1001sr BID as assessed by the NPSI and the BPI. Treatment with 80 mgTV1001sr BID also improved nerve function in diabetic neuropathypatients. These results indicate that administration of 80 mg TV1001srBID may be preferred for long-term treatment of pain and to improvenerve function in patients with chronic pain and reduced nerve function,such as patients having diabetic neuropathy.

OTHER EMBODIMENTS

Various modifications and variations of the described methods will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific embodiments, it will be understood that itis capable of further modifications and that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the art are intended tobe within the scope of the invention. This application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure come within known customarypractice within the art to which the invention pertains and may beapplied to the essential features herein before set forth.

1-53. (canceled)
 54. A method of treating or reducing pain in a patient,the method comprising: (i) administering between 5 mg to 50 mg of atherapeutic to the patient two times per day for a first treatmentperiod of 6 weeks to 14 weeks; and then (ii) administering 60 mg to 100mg of the therapeutic to the patient two times per day for a secondtreatment period of 6 weeks to 14 weeks; wherein the therapeutic is aninorganic nitrite or a pharmaceutically acceptable salt thereof.
 55. Themethod of claim 54, wherein the patient has a microvascular disease. 56.The method of claim 54, further comprising determining whether thepatient exhibits a reduction in pain.
 57. The method of claim 56,wherein the reduction in pain is determined as a decrease in painintensity, frequency, duration, and/or improvements in quality of life.58. The method of claim 56, further comprising performing a Brief PainInventory, a Neuropathic Pain Symptom Inventory, and/or a McGill PainQuestionnaire to determine if the patient exhibits a reduction in pain.59. The method of claim 54, wherein step (i) of the method results in areduction in pain.
 60. The method of claim 39, wherein step (ii) isperformed after the patient exhibits a reduction in pain.
 61. The methodof claim 54, wherein the first treatment period is at least 7 weeks, atleast 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks,at least 12 weeks, or at least 13 weeks.
 62. The method of claim 54,wherein the first treatment period is at least 10 weeks; the secondtreatment period is one of at least 7 weeks, at least 8 weeks, at least9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, and atleast 13 weeks; the therapeutic is NaNO₂, and is formulated for one oftopical, enteral, parenteral administration, as a solid dosage form fororal administration, as a tablet or capsule formulated for sustainedrelease of the inorganic nitrite; the therapeutic is formulated with oneor more pharmaceutically acceptable excipients; the therapeutic ispresent in an amount of 40 mg in step (i) of the method; the therapeuticis present in an amount of 80 mg in step (ii) of the method; the patienthas type 1 diabetes or type 2 diabetes; the patient is a mammal; and thepatient is a human.
 63. A method of treating or reducing pain in apatient, the method comprising: (i) administering a first amount of atherapeutic to the patient two times per day for a first treatmentperiod; and then (ii) administering a second amount of the therapeuticto the patient two times per day for a second treatment period; whereinthe therapeutic is inorganic nitrite or a pharmaceutically acceptablesalt thereof; and the first amount is less than the second amount. 64.The method of claim 63, wherein the second amount is at least twice thefirst amount.
 65. The method of claim 63, wherein the first period isequal to the second period.
 66. The method of claim 63, wherein thefirst period is less than the second period.
 67. The method of claim 63,wherein the second period is one of at least 6 weeks, at least 7 weeks,at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11weeks, at least 12 weeks, at least 13 weeks, and at least 14 weeks. 68.The method of claim 63, wherein the inorganic nitrite is administered ina extended release formulation.
 69. The method of claim 63, wherein theinorganic nitrite is one of ammonium nitrite (NH4NO2), barium nitrite(Ba(NO2)2; calcium nitrite (Ca(NO2)2; cesium nitrite (CsNO2), cobalt(II)nitrite (Co(NO2)2), cobalt(III) potassium nitrite (CoK3(NO2)6; lithiumnitrite (LiNO2); magnesium nitrite (MgNO2); potassium nitrite (KNO2),rubidium nitrite (RbNO2), silver (I) nitrite (AgNO2), sodium nitrite(NaNO2), strontium nitrite (Sr(NO2)2), and zinc nitrite (Zn(NO2)2). 70.The method of claim 63, wherein the first amount is between 30 mg and 50mg and the second amount is between 70 mg and 85 mg.
 71. A method oftreating or reducing pain in a patient, the method comprising: (i)administering between 30 mg and 50 mg of a therapeutic to the patienttwo times per day for a first treatment period; and then (ii)administering between 70 mg and 85 mg of the therapeutic to the patienttwo times per day for a second treatment period; wherein the therapeuticis NaNO2 or a pharmaceutically acceptable salt thereof; the first periodis less than the second period; the second period is one of at least 7weeks; and the therapeutic is administered via for oral administration,as a tablet or capsule formulated for sustained release, and isformulated with one or more pharmaceutically acceptable excipients.